The pluripotent stem cells capable of infinitely proliferating without causing cancerization and the like and having pluripotency have been expected to be applied to cell transplantation treatment, drug discovery screening, and the like.
Heretofore, a human pluripotent stem cell line has been proliferated and maintained by plate culture of causing the human pluripotent stem cell line to adhere to feeder cells, various polymers, or the like. However, a technique of stably culturing a large amount of high quality human pluripotent stem cells has not been established yet. In particular, a former method including causing the cell line to adhere to a culture container, and then proliferating the same by subculturing has been insufficient for preparing a large amount of pluripotent stem cells which are necessary for practical use. For example, the optimal adhesion substrate material for human pluripotent stem cells in terms of quality and cost has not been developed. Moreover, complicated many stages have been necessary for the subculturing, and thus disadvantageous steps in terms of safety and cost, such as enzyme treatment, have been included.
Recently, a suspension culture method which does not require an adhesion substrate has been reported (Patent Literatures 1 to 5). According to the suspension culture method, three-dimensional culture can be performed, and therefore a large amount of pluripotent stem cells can be cultured in a smaller space.
As a method for substituting the enzyme treatment in subculturing, a method including causing a cell mass of pluripotent stem cells to pass through a micro grid for dividing the cell mass has been devised (Patent Literature 6).